McGee W A, Nall B T
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Protein Sci. 1998 May;7(5):1071-82. doi: 10.1002/pro.5560070501.
N52I iso-2 cytochrome c is a variant of yeast iso-2 cytochrome c in which asparagine substitutes for isoleucine 52 in an alpha helical segment composed of residues 49-56. The N52I substitution results in a significant increase in both stability and cooperativity of equilibrium unfolding, and acts as a "global suppressor" of destabilizing mutations. The equilibrium m-value for denaturant-induced unfolding of N52I iso-2 increases by 30%, a surprisingly large amount for a single residue substitution. The folding/unfolding kinetics for N52I iso-2 have been measured by stopped-flow mixing and by manual mixing, and are compared to the kinetics of folding/unfolding of wild-type protein, iso-2 cytochrome c. The results show that the observable folding rate and the guanidine hydrochloride dependence of the folding rate are the same for iso-2 and N52I iso-2, despite the greater thermodynamic stability of N52I iso-2. Thus, there is no linear free-energy relationship between mutation-induced changes in stability and observable refolding rates. However, for N52I iso-2 the unfolding rate is slower and the guanidine hydrochloride dependence of the unfolding rate is smaller than for iso-2. The differences in the denaturant dependence of the unfolding rates suggest that the N52I substitution decreases the change in the solvent accessible hydrophobic surface between the native state and the transition state. Two aspects of the results are inconsistent with a two-state folding/unfolding mechanism and imply the presence of folding intermediates: (1) observable refolding rate constants calculated from the two-state mechanism by combining equilibrium data and unfolding rate measurements deviate from the observed refolding rate constants; (2) kinetically unresolved signal changes ("burst phase") are observed for both N52I iso-2 and iso-2 refolding. The "burst phase" amplitude is larger for N52I iso-2 than for iso-2, suggesting that the intermediates formed during the "burst phase" are stabilized by the N52I substitution.
N52I异-2细胞色素c是酵母异-2细胞色素c的一种变体，在由49 - 56位残基组成的α螺旋片段中，异亮氨酸52被天冬酰胺取代。N52I取代导致平衡去折叠的稳定性和协同性显著增加，并作为不稳定突变的“全局抑制因子”。变性剂诱导N52I异-2去折叠的平衡m值增加30%，对于单个残基取代来说，这是一个惊人的大数值。已通过停流混合和手动混合测量了N52I异-2的折叠/去折叠动力学，并与野生型蛋白异-2细胞色素c的折叠/去折叠动力学进行了比较。结果表明，尽管N52I异-2具有更高的热力学稳定性，但异-2和N52I异-2的可观测折叠速率以及折叠速率对盐酸胍的依赖性是相同的。因此，突变诱导的稳定性变化与可观测的重折叠速率之间不存在线性自由能关系。然而，对于N52I异-2，去折叠速率较慢，且去折叠速率对盐酸胍的依赖性比异-2小。去折叠速率对变性剂依赖性的差异表明，N52I取代减少了天然态和过渡态之间溶剂可及疏水表面的变化。结果的两个方面与两态折叠/去折叠机制不一致，并暗示存在折叠中间体：(1) 通过结合平衡数据和去折叠速率测量，由两态机制计算出的可观测重折叠速率常数偏离了观测到的重折叠速率常数；(2) 在N52I异-2和异-2重折叠过程中均观察到动力学上未解析的信号变化（“爆发相”）。N52I异-2的“爆发相”幅度比异-2大，表明“爆发相”期间形成的中间体因N52I取代而稳定。
